1. Field of the Invention
The present invention generally relates to compositions and methods for culturing pluripotent stem cells and/or primary tumor cells comprising an Erbb2 variant, the cells created by these methods, and the uses thereof. Particularly, the invention relates to the identification of cancer stem cell specific markers. In addition, the invention relates to the use of a modified defined culture medium for producing cultures of cancer stem cells in the absence of a feeder cell layer, in the absence of serum or serum replacement, and independent of heregulin.
2. Background Art
The process of embryonic development establishes the differentiated lineages of the body and sets aside tissue specific progenitor cells, which are also called stem cells. These progenitor cells are capable of regenerating all the relevant lineages of individual tissues during normal cellular turnover, or after injury. Examples of this regeneration include the constant regeneration of the skin, and reconstitution of the hematopoetic system following transplantation of hematopoetic stem cells. These progenitor cells typically reside in a “stem cell niche” and are relatively long lived as compared to their differentiated progeny. Embryonic stem (ES) cells represent a powerful model system for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo, as well as providing opportunities for genetic manipulation of mammals and resultant commercial, medical, and agricultural applications. Furthermore, appropriate proliferation and differentiation of ES cells can be used to generate an unlimited source of cells suited to study cell differentiation and/or suited for transplantation for treatment of diseases that result from cell damage or dysfunction.
Accumulated genetic change leading to unregulated cell growth is a hallmark of cancer progression. Because of their longevity, tissue specific stem cells may have a greater chance of accumulating mutations than differentiated cells, which exhibit comparatively rapid turnover in many tissues. Because tissue specific stem cells are the progenitor cells of other cells within that tissue, deregulation of the balance between quiescence, self-renewal, differentiation, or apoptosis of the tissue specific stem cells can have severe consequences. Any increase in the proliferation of these cells could lead to a magnified over-proliferation of downstream cell populations, leading to tumorigenesis. Furthermore, should mutations enable maintenance or expansion of these progenitor cells, self-perpetuating “cancer stem cells” (CSC) could be generated within tumors. It has long been known that a relatively small proportion of cells within differentiated tumors have the capacity to regenerate tumors at high frequency after transplantation (See, e.g., U.S. Pat. No. 6,984,522). This is indicative of rare populations of transformed cells that have the capacity to expand and differentiate to all lineages of the tumor. There is a need in the art for a natural model system in which to study the development of and signaling within a cancer stem cell population.
Human embryonic stem cells (hESCs) are pluripotent cells that can be isolated from the human blastocyst and maintained in culture as self-renewing cells, or differentiated to representatives of all mature tissues in the body. hESCs may therefore be a valuable model of human development in vitro, and are the focus of substantial research aimed at generating differentiated populations for cellular therapies. hESCs are “master” stem cells and theoretically capable of differentiating to all lineages, including tissue specific progenitor cells. Because hESCs are a self-renewing progenitor population, they are likely to use growth factors and molecular signaling pathways that are overlapping with some tissue specific stem cells and their related CSCs.
The successful isolation, long-term clonal maintenance, genetic manipulation, and germ-line transmission of pluripotent cells has generally been difficult, and the biochemical mechanisms regulating ES cell pluripotency and differentiation are very poorly understood. However, the limited empirical data available and much anecdotal evidence suggest that the continued maintenance of pluripotent ES cells under in vitro culture conditions is dependent upon the presence of cytokines and growth factors present in the extracellular milieu. Until recently, the standard growth conditions for hESCs were relatively undefined. The inventors recently developed a simple media for HESC growth that has several advantages over other reported defined media. The media relies on growth factor signaling through the EGF receptor family of cell surface proteins to maintain hESC pluripotency. This signaling is primarily transmitted through the Erbb2 receptor, a strong activator of the PI3 kinase pathway when present as heterodimers with other Erbb receptor family members. Four growth factors (bFGF, TGF-I, Activin A, and Heregulin) were required to inhibit spontaneous differentiation and promote self renewal of hESCs in this defined media. This system enabled the robust expansion of hFSCs and facilitated the examination of such cells in a standardized and simple background.
Neuregulin-1 (NRG1; heregulin) is a large gene that exhibits multiple splicing and protein processing variants. This generates a large number of protein isoforms, which are referred to herein collectively as neuregulin. Neuregulin is predominantly expressed as a cell surface transmembrane protein. The extracellular region contains an immunoglobulin-like domain, a carbohydrate modified region and the EGF domain. NRG1 expression isoforms have been reviewed previously (Falls, 2003, Exp. Cell Res., 284:14-30). The cell membrane metalloproteases ADAM17 and ADAM19 have been shown to process the transmembrane form(s) of neuregulin-1 to soluble neuregulin/heregulin. HRG-α and -β are the cleaved ectodomains of neuregulin, containing the EGF and other domains. As the EGF domain is responsible for binding and activation of the Erbb receptors, a recombinant molecule containing only this domain can exhibit essentially all of the soluble growth factor effects of this protein (Jones et al., 1999, FEBS Lett., 447:227-231). Also, there are processed transmembrane isoforms of neuregulin that are thought to trigger juxtacrine signaling in adjacent cells via interaction of the EGF domain with Erbb receptors.
The EGF growth factor family has at least 14 members, including, but not limited to, EGF, TGFα, heparin binding-EGF (hb-EGF), neuregulin-β (also named heregulin-β (HRG-β), glial growth factor and others), HRG-α, amphiregulin, betacellulin, and epiregulin. All these growth factors contain an EGF domain and are typically first expressed as transmembrane proteins that are processed by metalloproteinase (specifically, ADAMs) proteins to generate soluble ectodomain growth factors. EGF family members interact with both homo- and hetero-dimers of the Erbb1, 2, 3, and 4 cell surface receptors with different affinities (Jones et at, 1999, FEBS Lett, 447:227-231). EGF, TGFα, and hbEGF bind Erbb1/1 (EGFR) homodimers and Erbb1/2 heterodimers at high affinity (1-100 nM range), whereas HRG-β binds Erbb2/3 and Erbb2/4 heterodimers at very high affinity (<1 nM range). Activated Erbb receptors signal through the PI3 Kinase/AKT pathway, the MAPK pathway, and several other pathways (Oda et al., 2005, Mol. Sys. Biol., 1:2005.0010. Epub May 25).
Erbb2 and Erbb3 are amongst the most highly expressed growth factor receptors in hESCs (Sperger et al., 2003, PNAS, 100(23):13350-13355), and HRG-β has been shown previously to support the expansion of mouse primordial germ cells (Toyoda-Ohno et al., 1999, Dev. Biol., 215(2):399-406). Furthermore, over-expression and subsequent inappropriate activation of Erbb2 is associated with tumorigenesis (Neve et al., 2001, Ann. Oncol., 12 Suppl 1:S9-13; Zhou & Hung, 2003, Semin. Oncol., 30(5 Suppl 16):38-48; Yarden, 2001, Oncology, 61 Suppl 2:1-13). Human Erbb2 (Chromosome 17q between positions 11.2 and 12), and Erbb3 (Chromosome 12q13) are present on chromosomes that have been observed to accumulate as trisomies in some hESCs (Draper et al., 2004, Nat. Biotechnol., 22(1):53-4; Cowan et al., 2004, N Engl. J. Med., 350(13):1353-6; Brimble et al., 2004, Stem Cells Dev., 13(6):585-97; Maitra et al., 2005, Nat. Genet., 37(10):1099-103; Mitalipova et al., 2005, Nat. Biotechnol., 23(1):19-20; Draper et al., 2004, Stem Cells Dev., 13(4):325-36; Ludwig et al., 2006, Nature Biotechnol., 24(2):185-87), possibly suggesting that over-expression and/or activation of these receptors could be associated with the purported growth/survival advantage conferred by trisomies of these chromosomes.
The proto-oncogene Erbb2 is known in the art by several additional names, including, among others, human epidermal growth factor receptor 2 (HER2), C-erbB-2, ERB2_HUMAN, Her-2/neu, MLN 19, NEU, NEU proto-oncogene, NGL, Oncogene NGL, neuroblastoma- or glioblastoma-derived, p185erbB2, TKR1, Tyrosine kinase-type cell surface receptor HER2, V-Erb-B2, and Oncogene Erbb2. Various groups identified the genes and/or mapped the location to the long arm q of Chromosome 17 (Coussens et al., 1985, Science, 230:1132-1139; Semba et al., 1985, Proc. Nat. Acad. Sci., 82:6497-6501; Yang-Feng et al., 1985, Cytogenet. Cell Genet., 40:784; Di Fiore et al., 1987, Science, 237:178-182; Fukushige et al., 1986, Mol. Cell. Biol., 6:955-958; Kaneko et al., 1987, Jpn. J. Cancer Res., 78:16-19; Popescu et alt, 1989, Genomics, 4:362-366; Anderson et al., 1993, Genomics, 17:618-623; Muleris et al., 1997, Cytogenet. Cell Genet., 76:34-35). The Erbb2 gene consists of 27 exons, and the mRNA is approximately 3,768 nucleotides. Akiyama et al. (1986, Science 232:1644-46) raised antibodies against a synthetic peptide corresponding to 14 amino acid residues at the COOH terminus of the predicted Erbb2 protein, and they immunoprecipitated an 185-kD Erbb2 glycoprotein with tyrosine kinase activity from adenocarcinoma cells. The Erbb2 protein consists of 1255 amino acids. Erbb2 is a tyrosine kinase with a single transmembrane domain that separates an intracellular kinase domain from an extracellular domain. Erbb2 protein is expressed in several human organs and tissues, including normal epithelium, endometrium, ovarian epithelium, prostate, pancreas, lung, kidney, liver, heart, and hematopoietic cells. Erbb2 plays a role in normal development and differentiation.
Several researchers have identified the over-expression of Erbb2 as being involved in various cancers and have analyzed Erbb2's role as an oncogene. For example, increased expression of Erbb2 was noted in a human adenocarcinoma of the salivary gland (Semba et al., 1985, Proc. Nat. Acad. Sci., 82:6497-6501), a gastric cancer cell line (Fukushige et al., 1986, Mol. Cell. Biol., 6:955-958), a large-cell, comedo growth type of ductal carcinoma (Van de Vijver et al., 1988, New Eng. J. Med., 319:1239-1245), breast and ovarian cancer (Slamon et al., 1989, Science, 244:707-71 2; Yu et al., 1998, Mol. Cell, 2:581-591; Kun et al., 2003, Hum. Mol. Genet. 12:3245-3258; Menendez et al., 2004, Proc. Nat. Acad. Sci., 101:10715-10720), prostate cancer (Qiu et al., 1998, Nature, 393:83-85), acute lymphoblastic leukemia, bladder cancer, cervical cancer, childhood medulloblastoma, colorectal cancer, oral squamous cell carcinoma, germ-cell testicular cancer, cholangiocarcinoma, lung cancer, osteosarcoma, pancreatic adenocarcinoma, primary fallopian tube carcinoma, and synovial sarcoma. Di Fiore et al. (1987, Science, 237:178-182) demonstrated that over-expression alone can convert the Erbb2 gene into an oncogene. Other researchers have suggested that levels of Erbb2 expression could be used in determining the prognosis and/or chemosensitivity of human cancers, especially breast and ovarian cancer (Pegram et al., 1997, Oncogene, 15:537-547; Mehta et al., 1998, Oncol., 16:2409-2416; De Placido et al., 1998, Breast Cancer Res. Treat., 52:55-64). Erbb2 over-expression has been reported in 30-50% of ovarian carcinomas and is associated with advanced disease stage, worse prognosis, and decreased response to therapy in ovarian carcinoma patients; however, the molecular mechanisms underlying Erbb2 oncogenic activities in human cancer are unclear. Erbb2 over-expression is thought to be the mechanism of Erbb2 activation in certain cancers.
Other researchers have attempted to elucidate the function and activity of Erbb2. Qiu et al. (1998, Nature, 393:83-85) showed that Erbb2 forms a complex with the gp130 subunit of the IL6 receptor (IL6R) in an IL6-dependent manner and that Erbb2 is a critical component of IL6 signaling through the MAP kinase pathway. Yu et al. (1998, Mol. Cell, 2:581-591) found that over-expression of Erbb2 inhibits Taxol-induced apoptosis in breast cancers. The resistance to taxol-induced apoptosis is thought to be through the inhibition of p34 (CDC2) activation, via Erbb2-mediated upregulation of p21(CIP1), or CDKN1A, which inhibits CDC2. Tan et al. (2002, Mol. Cell, 9:993-1004) reported that the inhibitory phosphorylation on tyr15 (Y15) of CDC2 was elevated in Erbb2-overexpressing breast cancer cells and primary tumors, and concluded that Erbb2 can confer resistance to taxol-induced apoptosis by directly phosphorylating CDC2. In addition, Menendez et al. (2004, Proc. Nat. Acad. Sci., 101:10715-10720) identified a molecular link between the biosynthetic enzyme fatty acid synthase (FASN), which is associated with more aggressive breast and ovarian cancers, and the Erbb2 oncogene. Pharmacologic and RNAi FASN inhibitors were found to suppress Erbb2 expression and tyrosine kinase activity in breast and ovarian cancers over-expressing Erbb2.
Certain mutations and polymorphisms in the Erbb2 protein have been identified as having an association with breast cancer, adenocarcinoma, glioblastoma, gastric cancer, and ovarian carcinoma (die et al., 2000, J. Nat. Cancer Inst., 92:412-417; The Cancer Genome Project and Collaborative Group, 2004). Alternative splicing results in several additional transcript variants, some encoding different isoforms and others that have not been fully characterized. Doherty et al. (1999, Proc. Nat. Acad. Sci., 96:10869-10874) described a secreted protein of approximately 68 kDa, designated herstatin, as the product of an alternative Erbb2 transcript that retains intron 8. Herstatin appears to be an inhibitor of p185Erbb2, because it disrupts dimers, reduces tyrosine phosphorylation of p185, and inhibits the anchorage-independent growth of transformed cells that over-express Erbb2.
Another Erbb2 splice variant (referred to here as Erbb2Δ16) has been identified which harbors a deletion of exon 16, resulting in a 16 amino acid in-frame deletion in a small extracellular region of wild type Erbb2 (Siegel et al., 1999, EMBO J., 18(8):2149-64; Castiglioni et al., 2006, Endocr. Relat. Cancer, 13(1):221-32). Erbb2Δ16 has been implicated as an oncogeneic isoform of Erbb2. Exon 16 also contains a cleavage site for ectodomain shedding of wild type Erbb2 (Yuan et al., 2003, Prot. Exp. Pur., 29:217:222), and shedding may, therefore, be substantially altered in Erbb2Δ16. Exon 16 also contains two cysteine residues that are usually involved in disulfide bonds with other regions of the molecule, and Erbb2Δ16 may therefore have a different structure than wild type Erbb2. Disulfide linked homodimers and/or heterodimers of Erbb2Δ16 may form in cells transfected with an Erbb2Δ16 expression construct. The region of the deletion in Erbb2Δ16 is also a hotspot for mutations in Erbb2 that lead to breast cancer in mouse and rat models. Some researchers have reported that this oncogenic Erbb2 splice variant has been detected in several breast cancer cell lines, but was not over-expressed in breast tumors.
Brumlik et al. (Poster, 2003, Faseb Summer Research Conference: Growth Factor Receptor Tyrosine Kinases in Mitogenesis. Omni-Tucson, Tucson, Ariz.) developed an RT-PCR assay to quantitate Erbb2Δ16 expression and have generated preliminary data that identified over-expression of Erbb2Δ16 in ovarian and breast cancer cell lines and primary ovarian tumors from patients with advanced disease, suggesting that Erbb2Δ16 may in fact contribute to disease progression and would therefore represent a viable target for therapeutic intervention of ovarian cancer. In addition, this group targeted a unique sequence at the Erbb2Δ16 exon 15 and exon 17 junction for suppression by RNA interference (RNAi) and demonstrated the efficacy and specificity of RNAi to suppress Erbb2Δ16 expression in ovarian cancer cells.
Similarly, Castiglioni et al. (2006, Endocrine Related Cancer, 13:221-232) have reported that the Erbb2Δ16 splice variant represents about 9% of the Erbb2 mRNA obtained from most of the 46 breast carcinoma samples in that study. They found that human cells transfected with wild type Erbb2 cDNA revealed no growth of wild type cells in nude mice, whereas clones expressing 10-fold less Erbb2Δ16 were tumorigenic. In addition, they noted that unlike wild type Erbb2-transfectants, Erbb2Δ16-expressing cells showed low sensitivity to two therapeutic drugs targeting receptors of the HER family (ZD1839 and Trastuzumab (Herceptin®), whereas an inhibitor of the HER2 tyrosine kinase domain (Emodin) blocked activation of both Erbb2Δ16 and wild type Erbb2 transfectants. They suggested that their data indicate that the Erbb2Δ16 transcript encodes the transforming form of the oncoprotein and that malignant transformation arises when a critical threshold of Erbb2Δ16 is reached in Erbb2 over-expressing tumors.
Therapies directed toward Erbb2 are currently being used effectively in breast cancer patients; however, these treatments have significant drawbacks. In particular, a humanized anti-Erbb2 monoclonal antibody, Herceptin® (Genentech), has been shown to be effective in slowing the progression of approximately 30% of breast cancers, demonstrating the role of this receptor in tumor growth. Slamon et al. (2001, Science, 244:707-712) found in a large-scale clinical trial that treatment with Herceptin® increased the clinical benefit of first-line chemotherapy in metastatic breast cancer that over-expresses Erbb2. Although Hercepti® demonstrated clinical efficacy, a significant number of women suffered from the severe side effect of treatment-induced cardiotoxicity.
In addition, small molecule inhibitors of the Erbb2 tyrosine kinase (TK) domain have been shown to effectively inhibit proliferation of breast cancer cells in culture and in animal models of tumor growth. The inventors also have shown that three different Erbb2 TK inhibitors, AG879, AG825, and emodin, inhibit the growth of BG01v cells in defined media, in the absence of an exogenous Erbb2 activating growth factor. This confirms that endogenous Erbb2 signaling is critical for the self-renewal of BG01v cells and represents a major difference between variant and normal hESCs.
What is needed in the art are model systems for studying the transformation of tissue progenitor cells to cancer stem cells and the elucidation of media and cell culture conditions that are capable of effecting such transformation. Also needed are new methods for the specific targeted treatment of breast cancer and other tumors. There is a need, therefore, to identify methods and compositions for the culture of a population of cancer stem cells that are able to be used for research purposes to study tumorigenesis. There is also a need to identify markers that are specific to the cancer stem cells that may be used as targets for therapeutic treatments of cancer and that may be used to facilitate the prognosis of patients with various tumors.